1. Field of the Invention
This invention relates to peptides possessing biological activity, particularly for use in vaccines for diseases caused by enterovirus. The invention further relates to anti-viral therapeutics, particularly recombinant human anti-EV71 monoclonal antibodies. The invention further relates to application of said antibodies for use in therapy, surgery and diagnosis of EV71 infection.
2. Description of Prior Art
Hand, foot and mouth disease (HFMD) is generally a mild, self-limited disease that primarily affects infants and young children. The infection of enterovirus 71 (EV71) is the second most common cause of HFMD (CDC report, Aug. 7, 1998; CDC report, Aug. 11, 1998). The infection of EV71 is often complicated with severe neurological manifestations including viral (aseptic) meningitis, encephalitis, and a polio-like paralysis.
EV71 was first isolated and characterized in 1969 (Schmidt et al., 1974, J. Infect. Dis. 129:304). Many reports of EV71 outbreaks were filed around the world before 1998, mostly in Southeast Asia (Lam, 1998, Emerg. Infect. Dis. 4:145; WHO report, June, 1998; WHO report, July, 1998). The World Health Organization had reported Hong-Kong, Malaysia, and Taiwan as epidemic areas. The most serious outbreak of all epidemics was the one in Taiwan during April-July 1998, resulting in about 320 cases with severe complications and at least 73 deaths (Wu et al., 1999, Emerg. Infect. Dis, 5:458; Wang et al., 1999, Clin. Infect. Dis. 29(1):184; Komatsu et al., 1999, Pediatr. Neurol. 20:17).
The period from infection of EV71 to the onset of symptoms in infants is 3 to 6 days. Fever is often the first symptom of HFMD. One or two days after the fever begins, sores develop in the mouth and often become ulcers later. The skin rash develops for 1 to 2 days, usually located on the palms of hands and soles of feet. Most patients recover in 1 to 2 weeks (WHO report, June, 1998; CDC report, Aug. 7, 1998). Some cases, mostly children younger than 3 years old, however, develop severe complications. They exhibited a short (2-4 days) febrile illness, followed by a sudden deterioration, and died within 12 to 24 hours (CDC report, Aug. 07, 1998). It is not clear why most infected children spontaneously recover, while some succumb to the infection and develop complication at a very fast course.
It has been reported that EV71-neutralizing IgM is produced in monkey 14 to 20 days after infection (Hashinmoto and Hagiwara, 1982, Neuropathol. Appl. Neurobiol. 8:149; Hashinmoto and Hagiwara, 1983, Acta. Neuropathol 60:266). It is suggested that the sudden death, which occurs at about day 7, of some infected children is due to their inability of producing sufficient neutralizing antibodies in time. Currently there is no effective agent or treatment to counter the infection of EV71. Available treatments of HFMD are often symptomatic, directed to relieving fever, headache, and malaise. Managing complications of the disease complications has become the most important concern in clinical caring (CDC report, Aug. 11, 1998). Therefore, an effective treatment to inhibit viral propagation and to clear virus particles is highly desirable.
The technique for producing monoclonal antibodies (mAbs) using the hybridoma methodology was invented in 1975 (Cottona and Milstein, 1973, Nature 244:42; Kohler and Milstein, 1975, Nature 256:495). In the past two decades, a number of mAbs for therapeutic applications have been developed. These mAbs recognize critical molecules or pathogens of a specific disease, and mediate immune mechanisms to eliminate them. Up to 2001, eleven murine gene-modified mAbs have reached the market, and 3 await regulatory approval (Glennie and Johnson, 2000, Immunol. Today 21:403; Ezzell, 2001, Sci. Am. 285:34).
Synagis is one of the successful monoclonal antibodies designed to recognize the virus-neutralizing site of respiratory syncytial virus (RSV), the F protein, and thus inhibit the RSV infection (Malley et al., 1998, J. Infect. Dis. 178:1555; Marchetti et al., 1999, Clin. Ther. 21:752.). Such notable success has stimulated wide academic and industrial interests of developing monoclonal antibodies as therapeutic agents. More than 100 mAbs are now being tested in human and have shown encouraging results.
EV71 is a positive single stranded RNA virus. The viral RNA is encapsidated by four structural proteins: viral protein 1 (VP1), VP2, VP3, and VP4, which are produced by post-translational cleavages of a common naked protein precursor. Of these viral proteins, VP1 has been suggested to be mainly responsible for attachment of enteroviruses to target cell (Graham et al., 1989, J. Gen. Virol. 70:625) and hence harbors the main antigenic determinants for virus neutralization (Pfaff et al., 1988, J. Virol. 62:2033; Rueckert, 1990, Virology/Lippin-cott-Raven, p. 507). Immunological studies with different enterovirus strains have indicated that the dominant epitopes of several monoclonal neutralizing antibodies (mAbs) are located on VP1 (Philip et al., 1986, J. Gene. Virol. 67:1283; Tapani et al., 1993, J. Clin. Microbiol. 31:1083; Hyppia et al., 1997, J. Gene. Virol. 78:1).
Several groups have identified short segments of VP1 as crucial elements in inducing protective immunity and have designed oligopeptides for possible applications (U.S. Pat. No. 4,694,071, U.S. Pat. No. 4,751,083, U.S. Pat. No. 4,875,643). Pregnant ICR mice immunized with VP1 proteins or DNA of EV71 have also been found to produce sufficient neutralizing antibodies to protect newborn mice from EV71 infection (Wu, C. N. et al., 2002, Vaccine 20:895).
Although EV71 is a genetically diverse, rapidly evolving virus, the 3 genotypes of VP1 proteins of all isolated strains from more than 6 countries, including the prototype strain BrCr-CA-70, are at least 94% identical to one another (Brown et al., 2000, J. Viol. 74:12003). During the outbreak of EV71 in Taiwan, many clinical isolates were obtained and characterized. It was found that the VP1 proteins found in different regions of Taiwan have almost identical sequences. Comparing amino acid sequences of VP1 in GenBank showed that VP1 proteins share high sequence similarity between different isolates (>96%) and more than 98.9% identity within the same genotype (Wang et al., 2002, J. Clin. Microbiol. 40:10; Shih et al., 2000, Virus Res. 68:127). These data indicate that VP1 is very conserved and does not undergo significant mutations in infected individuals. Thus EV71 is a potent neutralizing site of EV71 and a suitable antigenic target.
In the past 15 years, the methodology of generating antibody has been greatly improved with the advent of antibody engineering and transgenic technologies. A new generation of antibodies, fully human monoclonal antibodies, has been produced for clinical applications. The best known methods of developing human monoclonal antibodies include displaying human VH-VL single chain (scFv) libraries on phage surface, preparing antibodies in transgenic mice, harboring genes of human heavy chain/light chain immunoglobulins and producing antibodies with hybridomas made with human myeloma cells (Soderlind et al., 1993, Biotechnol. 11:503; Vaughan et al., 1998, Nat. Biotechnol. 16:535; Karpas et al., 2001, Proc. Natl. Acad. Sci. USA 98:1799). Experimental data shows that these mAbs have much reduced immunogenicity, longer half-lives and enhanced immune activities in human patients.
Recently, a high throughput generation/screening system of human antibody repertoire has been constructed in yeast (for example, U.S. Pat. No. 6,406,863, the disclosure of which is hereby incorporated, in its entirety, by reference). As compared with a phage displayed library, antibodies produced in yeast perform protein folding after being expressed, and should be more functionally relevant to human antibodies then those expressed on phage surface. In addition, screening for antibody-antigen interaction can be conveniently carried out in yeast using a yeast two-hybrid method, which could save much time than conventional strategies described above. Hence, the yeast two-hybrid system provides an efficient and economical way to screen for fully human antibodies
Another advantage of the yeast antibody library is the high incidence of homologous recombination in yeast. DNA shuffling between the VH and VL sequences further increases the complexity of the antibody library and also increases the binding affinity of selected scFv clones after maturation. Anti-EV71 neutralizing antibodies with higher affinity could be obtained via antibody maturation.